AKI: an increasingly recognized risk factor for CKD development and progression.

Acute kidney injury (AKI) is an increasing health burden with high morbidity and mortality rates worldwide. AKI is a risk factor for chronic kidney disease (CKD) development and progression to end stage renal disease (ESRD). Rapid action is required to find treatment options for AKI, plus to anticipate the development of CKD and other complications. Therefore, it is essential to understand the pathophysiology of AKI to CKD transition. Over the last several years, research has revealed maladaptive repair to be an interplay of cell death, endothelial dysfunction, tubular epithelial cell senescence, inflammatory processes and more-terminating in fibrosis. Various pathological mechanisms have been discovered and reveal targets for potential interventions. Furthermore, there have been clinical efforts measures for AKI prevention and progression including the development of novel biomarkers and prediction models. In this review, we provide an overview of pathophysiological mechanisms involved in kidney fibrosis. Furthermore, we discuss research gaps and promising therapeutic approaches for AKI to CKD progression.

Hypercalciuria and nephrolithiasis: Expanding the renal phenotype of Donnai-Barrow syndrome.

Whole exome sequencing detected novel likely pathogenic variants in LRP2 gene in 2 patients presenting with hearing and vision loss, and the Dent disease (DD) classical renal phenotype, that is, low molecular weight proteinuria (LMWP), hypercalciuria and nephrocalcinosis/nephrolithiasis. We propose that a subset of patients presenting as DD may represent unrecognized cases or mild forms of Donnai-Barrow/facio-oculo-acustico-renal (DB/FOAR) syndrome or be on the phenotypic continuum between the 2 conditions.

Diagnostic specificity of autoantibodies to M-type phospholipase A2 receptor (PLA2R) in differentiating idiopathic membranous nephropathy (IMN) from secondary forms and other glomerular diseases.

Autoantibody against phospholipase A2 receptor (anti-PLA2R) is a sensitive and specific biomarker of idiopathic membranous nephropathy (iMN), being found in approximately 70% of iMN patients and only occasionally in other glomerular diseases. However, whereas its diagnostic specificity vs. normal controls and other glomerulonephritides (GN) has been firmly established, its specificity vs. membranous nephropathy associated with various diseases (sMN) has given inconsistent results. The aim of our study was to evaluate the prevalence of anti-PLA2R antibodies in iMN in comparison with various control groups, including sMN. A total of 252 consecutive iMN patients, 184 pathological and 43 healthy controls were tested for anti-PLA2R antibody using indirect immunofluorescence (PLA2R IIFT, Euroimmun). Anti-PLA2R autoantibodies were detectable in 178/252 iMN patients, 1/80 primary GN, 0/72 secondary GN, 9/32 sMN and 0/43 healthy controls, with a diagnostic sensitivity of 70.6%. The diagnostic specificity of anti-PLA2R antibody vs. normal and pathological controls was 100 and 94.6% respectively. However, when the diagnostic specificity was calculated only vs. secondary forms of MN, it decreased considerably to 71.9%. Interestingly enough, 9 out of 10 anti-PLA2R positive patients in the disease control groups had membranous nephropathy associated with various diseases (7 cancer, 1 Crohn's disease, 1 scleroderma). In conclusion, anti-PLA2R positivity in a patient with MN, should not be considered sufficient to abstain from seeking a secondary cause, especially in patients with risk factors for neoplasia. The causal relationship between tumors and anti-PLA2R-induced MN remains to be established, as well as the possible mechanisms through which malignancies provoke autoimmunity.

Endothelial progenitor cell-derived microvesicles improve neovascularization in a murine model of hindlimb ischemia.

Paracrine mediators released from endothelial progenitor cells (EPCs) have been implicated in neoangiogenesis following ischemia. Recently, we demonstrated that microvesicles (MVs) derived from EPCs are able to activate an angiogenic program in quiescent endothelial cells by a horizontal transfer of RNA. In this study we aim to investigate whether EPC-derived MVs are able to induce neoangiogenesis and to enhance recovery in a murine model of hindlimb ischemia. Hindlimb ischemia was induced in severe combined immunodeficient (SCID) mice by ligation and resection of the left femoral artery and mice were treated with EPC-derived MVs (MVs), RNase-inactivated MVs (RnaseMVs), fibroblast-derived MVs or vehicle alone as control (CTL). Since MVs contained the angiogenic miR-126 and miR-296, we evaluated whether microRNAs may account for the angiogenic activities by treating mice with MVs obtained from DICER-knock-down EPC (DICER-MVs). The limb perfusion evaluated by laserdoppler analysis demonstrated that MVs significantly enhanced perfusion in respect to CTL (0.50±0.08 vs 0.39±0.03, p<0.05). After 7 days, immunohistochemical analyses on the gastrocnemius muscle of the ischemic hindlimb showed that MVs but not fibroblast-MVs significantly increased the capillary density in respect to CTL (MVs vs CTL: 24.7±10.3 vs 13.5±6, p<0.0001) and (fibroblast-MVs vs CTL: 10.2±3.4 vs 13.5±6, ns); RNaseMVs and DICER-MVs significantly reduced the effect of MVs (RNaseMVs vs CTL: 15.7±4.1 vs 13.5±6, ns) (MVs vs DICER-MVs 24.7±10.3 vs 18.1±5.8, p <0.05), suggesting a role of RNAs shuttled by MVs. Morphometric analysis confirmed that MVs enhanced limb perfusion and reduced injury. The results of the present study indicate that treatment with EPC-derived MVs improves neovascularization and favors regeneration in severe hindlimb ischemia induced in SCID mice. This suggests a possible use of EPCs-derived MVs for treatment of peripheral arterial disease.

Loss of nephrin expression in glomeruli of kidney-transplanted patients under m-TOR inhibitor therapy.

The development of proteinuria has been observed in kidney-transplanted patients on m-TOR inhibitor (m-TORi) treatment. Recent studies suggest that m-TORi(s) may alter the behavior and integrity of glomerular podocytes. We analyzed renal biopsies from kidney-transplanted patients and evaluated the expression of nephrin, a critical component of the glomerular slit-diaphragm. In a group of patients on 'de novo' m-TORi-treatment, the expression of nephrin within glomeruli was significantly reduced in all cases compared to pretransplant donor biopsies. Biopsies from control transplant patients not treated with m-TORi(s) failed to present a loss of nephrin. In a group of patients subsequently converted to m-TORi-treatment, a protocol biopsy performed before introduction of m-TORi was also available. The expression of nephrin in the pre-m-TORi biopsies was similar to that observed in the pretransplant donor biopsies but was significantly reduced after introduction of m-TORi(s). Proteinuria increased after the m-TORi inititiation in this group. However, in some cases proteinuria remained normal despite reduction of nephrin. In vitro, sirolimus downregulated nephrin expression by human podocytes. Our results suggest that m-TORi(s) may affect nephrin expression in kidney-transplanted patients, consistently with the observation in vitro on cultured podocytes.

Antiangiogenic and immunomodulatory effects of rapamycin on islet endothelium: relevance for islet transplantation.

Donor intra-islet endothelial cells contribute to neovascularization after transplantation. Several factors may interfere with this process and ultimately influence islet engraftment. Rapamycin, a central immunosuppressant in islet transplantation, is an mTOR inhibitor that has been shown to inhibit cancer angiogenesis. The aim of this study was to evaluate the effects of rapamycin on islet endothelium. Rapamycin inhibited the outgrowth of endothelial cells from freshly purified human islets and the formation of capillary-like structures in vitro and in vivo after subcutaneous injection within Matrigel plugs into SCID mice. Rapamycin decreased migration, proliferation and angiogenic properties of human and mouse islet-derived endothelial cell lines with appearance of apoptosis. The expression of angiogenesis-related factors VEGF, alphaVbeta3 integrin and thrombospondin-1 on islet endothelium was altered in the presence of rapamycin. On the other hand, rapamycin decreased the surface expression of molecules involved in immune processes such as ICAM-1 and CD40 and reduced the adhesion of T cells to islet endothelium. Our results suggest that rapamycin exerts dual effects on islet endothelium inducing a simultaneous inhibition of angiogenesis and a down-regulation of receptors involved in lymphocyte adhesion and activation.

The expression of CD154 by Kaposi's sarcoma cells mediates the anti-apoptotic and migratory effects of HIV-1-TAT protein.

Kaposi's sarcoma (KS) is a malignancy associated to conditions of immune system impairment such as HIV-1 infection and post-transplantation therapy. Here we report that HIV-1-Tat protein, at concentrations well below those detected in AIDS patients, up-regulates the expression of both CD40 and CD154 on KS cells. This occurred also in the presence of vincristine, that at doses shown to induce apoptosis decreased the expression of both CD40 and CD154 on KS cells. The treatment with a soluble CD40-muIg fusion protein (CD40 fp) that prevents the binding of CD154 with cell surface CD40, as well as the transfection with a vector for soluble CD40 (KS sCD40), decreased the anti-apoptotic effect of Tat. Moreover, Tat-induced motility of KS cells was inhibited by soluble CD40 fp. Tat also enhanced the expression of intracellular proteins known to transduce signals triggered by CD40 engagement, in particular TRAF-3. Tat as well as soluble CD154 (sCD154) prevented vincristine-induced reduction of TRAF-3 in KS cells transfected with a vector for neomycin resistance (KS psv-neo), but not in KS sCD40. Immunoprecipitation studies showed that Tat induced CD40 / TRAF-3 association and that this binding was abrogated upon the incubation with the soluble CD40 fp. These data suggest that Tat activates the CD40-CD154 pathway by enhancing the membrane expression of CD40 and in particular of CD154, and by activating the TRAF-3-dependent signaling pathway of CD40. These findings indicate that the CD40-CD154 pathway mediates the anti-apoptotic and migratory effects of HIV-1- Tat, suggesting the potential therapeutic benefits of blocking CD40 activation in HIV-1-associated KS.

HIV type 1 Tat protein is a survival factor for Kaposi's sarcoma and endothelial cells.

The HIV-1 Tat protein has been directly implicated in the pathogenesis of AIDS-related Kaposi's sarcoma (KS); however, its effects on KS spindle-shaped and endothelial cell apoptosis are largely unexplored. Since susceptibility to apoptosis is relevant for tumor development and response to therapy, we investigated the effects of Tat on KS and endothelial cell survival from apoptosis. The effect of Tat was evaluated in three KS cell lines (KS-imm, KS-C1, and KS-L3) exposed to the chemotherapy agent vincristine, currently used for the treatment of this tumor, and in human umbilical vein-derived endothelial cells (HUVECs) induced to undergo apoptosis by serum withdrawal. Apoptosis was assessed by enzymatic assays, microscopic examination of chromatin and cytoskeleton, evaluation of plasma membrane integrity and subdiploid DNA content, TUNEL assays, and measurement of caspase-3 activity. Tat, in a dose-dependent manner, protected the three KS cell lines and HUVECs from apoptosis induced by vincristine or serum starvation, respectively. This effect appeared to be independent of modulation of Fas, Bcl-2, or Bax expression. In contrast, Tat upregulated Bcl-X(L) expression and induced a relevant decrease in caspase-3 activity in vincristine-treated KS cells. Taken together, these results suggest that the HIV-1 Tat protein may factor KS development and progression by sustaining endothelial and transformed cell survival.

Role of platelet-activating factor in functional alterations induced by xenoreactive antibodies in porcine endothelial cells.

BACKGROUND: Platelet-activating factor (PAF) is a phospholipid mediator of inflammation which has been implicated in rejection. The interaction of anti-alpha-galactosyl natural antibodies (anti-alpha gal Abs) with endothelial cells is the initial step for the development of xenograft rejection. In our study, we stimulated porcine aortic endothelial cells (PAEC) with anti-alpha gal IgG to investigate the synthesis of PAF from PAEC and its biological consequences. METHODS AND RESULTS: PAF was extracted and chromatographically purified from cultured PAEC stimulated with baboon anti-alpha gal Abs. The Abs induced a dose-dependent synthesis of PAF peaking after 30 min of incubation, and decreasing thereafter. Concomitant cell shape change, motility, and cytoskeleton redistribution were observed. These events were prevented by addition of a panel of PAF-receptor antagonists. An SV40 T-large antigen-immortalized PAEC line was engineered to express PAF acetyl-hydrolase (PAF-AH) cDNA, the major PAF-inactivating enzyme. These transfected cells exposed to anti-alpha gal Abs showed reduced cell contraction and motility compared with empty vector-transfected cells. Moreover, in PAEC stimulated with anti-alpha gal Abs, the synthesis of PAF promoted the adhesion of a monocytic cell line as shown by the inhibitory effect of PAF-receptor antagonists and of PAF-AH expression. Finally, studies on cell monolayer demonstrated an enhanced permeability 48 hr after exposure to anti-alpha gal Abs, and this increase was prevented by PAF-inactivation and by PAF-receptor blockade. CONCLUSIONS: These results demonstrate that on stimulation with anti-alpha gal Abs, PAEC synthetize PAF which can contribute to several vascular events involved in xenograft rejection.

Effect of platelet-activating factor receptor expression on CHO cell motility.

Tumor cell migration may favor local mass expansion and metastasis dissemination. Several tumors were found to express the receptor for platelet-activating factor (PAF), a potent mediator of leukocyte chemotaxis and endothelial cell migration. However, its functional role on tumor cells is largely unexplored. In the present study, we evaluated the motogenic effect of PAF on Chinese hamster ovarian (CHO) cancer cells transfected with the human PAF-receptor cDNA (CHO PAF-R). By using time-lapse recording, we detected a rapid motogenic response to PAF stimulation on CHO PAF-R, whereas no effect was evident on vector-only transfected cells. Such an effect was observed on scattered cell motility, on cells seeded on a fibronectin- or collagen-coated surface, and on migration of confluent monolayer cells. Cell speed increased at 1 h and was maximal 6-8 h after PAF stimulation on CHO PAF-R. Concomitantly, PAF induced marked changes in cytoskeleton actin distribution with cell contraction, assembling of stress fibers, and polar foci of adhesion. In conclusion, the present study demonstrates that PAF is a potent inducer of tumor cell motility, thus suggesting a role for this mediator in tumor growth and dissemination.

Motility induced by human immunodeficiency virus-1 Tat on Kaposi's sarcoma cells requires platelet-activating factor synthesis.

In the present study, we evaluated whether motility of Kaposi's sarcoma (KS) spindle cells induced by HIV-1 Tat protein is dependent on the synthesis of platelet-activating factor (PAF). The results obtained indicate that Tat induced a dose-dependent synthesis of PAF from KS cells at a concentration as low as 0.1 ng/ml. PAF production started rapidly after Tat stimulation, peaking at 30 minutes and declining thereafter. Tat-induced cell migration was also a rapid event starting at 30 minutes. The motility was abrogated by addition of a panel of chemically unrelated PAF receptor antagonists (WEB 2170, CV 3988, CV 6209, and BN 52021), suggesting that the synthesized PAF mediates the motogenic effect of Tat. This effect was also present on cells plated on a type-I collagen-, fibronectin-, or basement membrane extract-coated surface. Expression of PAF receptor-specific mRNA was detected in KS cells. In addition, examination of the cytoskeletal organization showed that Tat-mediated KS cell redistribution of actin filaments and shape change was also inhibited by a PAF receptor antagonist. Moreover, PAF receptor blockade prevented the up-regulation of beta1 integrin and the down-regulation of alphavbeta3 observed after stimulation of KS cells with Tat. In conclusion, the results of the present study indicate that Tat-induced PAF synthesis plays a critical role in triggering the events involved in motility of KS cells.

Activation of CD40 favors the growth and vascularization of Kaposi's sarcoma.

Although CD40 is expressed by several tumor lines and is up-regulated in tumor vascular endothelium, its role in tumor biology is still unclear. In the present study, we investigated the role of CD40 in the growth and vascularization of Kaposi's sarcoma (KS). In vitro, stimulation of CD40 induced migration of KS cells and inhibited vincristine-induced apoptosis. Similarly, the CD40 engagement on endothelial cells resulted in cell contraction, migration, and prevention of serum withdrawal-apoptosis. To understand the biological relevance of CD40 in vivo, KS cells were engineered to express and release a soluble form of CD40 (KS-sCD40) able to disrupt CD40-CD154 interaction. SCID mice s.c. injected with KS-sCD40 cells developed tumors that were significantly smaller than those induced by control cells (KS-neo). In addition, KS-sCD40 tumors showed several areas of necrosis, diffuse presence of apoptotic cells, and poor vascularization. In contrast, KS-neo tumors showed few or absent areas of necrosis and apoptosis and intense vascularization. Moreover, anti-CD40 Abs stimulated neo-angiogenesis in a murine model in which s.c. implantation of Matrigel was used as a vehicle for the delivery of mediators. These observations provide demonstration that CD40 supports tumor cell survival, growth, and neo-vascularization of KS.

Expression of L-selectin ligands by transformed endothelial cells enhances T cell-mediated rejection.

Recent immunohistochemical studies have suggested that L-selectin ligands may be implicated in the infiltration of tumors and rejected transplants by lymphocytes. In the present study, polyoma-middle T Ag-transformed endothelial cells (H.end), which typically form in vivo immunogenic vascular tumors resembling Kaposi's sarcoma, were engineered to express L-selectin ligands by stable transfection with a cDNA encoding alpha(1,3/4)-fucosyltransferase (H.endft). The ability of these cells to form tumors in the s.c. tissues of normal and immunocompromised mice was then compared with that of H.end cells transfected with the hygromycin-resistance vector only (H. endhygro). H.endhygro cells rapidly formed local and metastatic tumors in normal syngeneic mice, leading to death within 2-3 mo postinjection. By contrast, tumors derived from H.endft cells displayed a slower rate of growth, an absence of metastasis, and marked lymphocyte infiltration. Animals bearing these tumors survived for a significantly longer duration than animals injected with H.endhygro cells. Alternatively, H.endft and H.endhygro cells formed tumors with comparable aggressiveness in immunocompromised mice, resulting in animal death within 3 wk of injection. H.endft but not H.endhygro cells supported L-selectin-dependent adhesion and cytolytic T cell activity in vitro. Taken together, our observations indicate that the in situ expression of fucosyltransferase may significantly influence the cellular immune response in endothelioma tumors. These results may be relevant in understanding the development of vascular opportunistic tumors such as Kaposi's sarcoma.

CD40-CD154 interaction in experimental and human disease (review).

Cell-to-cell signals between T lymphocytes and antigen-presenting cells strictly regulate the development of the immune response. It has clearly emerged that among these signals few cell surface receptor-ligand pairs, such as CD40 and its ligand, CD154, are mandatory for the induction of lymphocyte activation. The early observation that mutations of CD154 gene are responsible for a human severe immunodeficiency primed an impressive number of studies aimed to functionally characterize this receptorial system in view of therapeutically exploiting its properties. Indeed, various approaches aimed to disrupt natural CD40-CD154 interaction were highly effective in the prevention and treatment of several experimental models of autoimmune disease and transplant rejection. In parallel, abnormalities of this pathway were constantly found in several immunologically-mediated human diseases. Furthermore, a number of studies have dissected the role of CD40 and its ligand in the immune response against various microbial and viral pathogens. Since these molecules are often expressed by tumor cells, it is not surprising that great efforts have been made to address their function also in the development of cancer. Most recent data strongly suggest an involvement of endothelial CD40 in the vascular processes that lead to atherogenesis. This review focuses on the most significant advances in the understanding of the molecular regulatory events involving CD40 and its ligand in experimental and human disease.